The overall goal of this project is to determine the mechanism by which the amino acid sequence of a protein directs the rapid and efficient folding and association required to produce multisubunit proteins. Specific aims involve biophysical studies of the folding mechanisms of two dimeric systems: the GCN4-p1 leucine zipper peptide and the tryptophan repressor (TR). The simple, coiled-coil structure of the GCN4-p1 system contrasts sharply with the intimately intertwined structure of TR. Previous equilibrium studies of both polypeptides show that only the native dimers and unfolded monomers are stable at equilibrium. Kinetic studies show that monomeric and dimeric intermediates appear during the folding of TR while GCN4-p1 appears to fold without populating intermediates. Calorimetric, optical, and NMR studies of mutant versions of these two systems will reveal thermodynamic, structural, and dynamic properties of stable states. Kinetic studies will probe transient intermediates in TR and mutational analysis will test the roles of various side chains in the rate limiting transition states for both proteins. Fragmentation of TR will highlight the behavior of an independently folding, dimeric core domain while chemical modification will provide access to a stable, monomeric form whose folding properties will also be examined. The information obtained should elucidate the molecular events which accompany the folding and association of subunits in these dimeric polypeptides. Advances in understanding the mechanism of folding of multisubunit proteins will have a significant impact on biochemistry, medicine, and the biotechnology industry.